Identification is proceeding for the new tRNA thionucleotide produced after bacteriophage lambda infection of Escherichia coli. Methods have been devised for large-scale isolation of the thionucleotide. The lambda site coding for the new thiolating activity has been mapped. An amber mutation at this site can be suppressed by tyrosine, but not by serine or glutamic acid substitutions. A search for further E. coli mutants deficient in tRNA 4-thiouridine has thus far been fruitless.